Structural mechanism of human oncochannel TRPV6 inhibition by the natural phytoestrogen genistein

Calcium-selective oncochannel TRPV6 is the major driver of cell proliferation in human cancers. While significant effort has been invested in the development of synthetic TRPV6 inhibitors, natural channel blockers have been largely neglected. Here we report the structure of human TRPV6 in complex with the plant-derived phytoestrogen genistein, extracted from Styphnolobium japonicum, that was shown to inhibit cell invasion and metastasis in cancer clinical trials. Despite the pharmacological value, the molecular mechanism of TRPV6 inhibition by genistein has remained enigmatic. We use cryo-EM combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to show that genistein binds in the intracellular half of the TRPV6 pore and acts as an ion channel blocker and gating modifier. Genistein binding to the open channel causes pore closure and a two-fold symmetrical conformational rearrangement in the S4–S5 and S6-TRP helix regions. The unprecedented mechanism of TRPV6 inhibition by genistein uncovers new possibilities in structure-based drug design.

a Representative micrograph with example particles circled in pink. A total of 3,630 such micrographs were collected, manually inspected and those with outliers in defocus values, ice thickness, and astigmatism as well as those with lower predicted CTF-correlated resolution (higher than 5 Å) were excluded (individually assessed for each parameter relative to the overall distribution), with 3,166 micrographs left for further processing (see Methods and Supplementary  Fig. 2). b,c Representative 2D class averages for hTRPV6Open (b) and hTRPV6GEN (c). d,e FSC curves for hTRPV6Open (d) and hTRPV6GEN (e). f,g Euler angle distribution of particles contributing to final reconstructions of hTRPV6Open (f) and hTRPV6GEN (g) with larger red cylinders representing orientations comprising more particles. h,i Local resolution presented as coloring of the cryo-EM maps of hTRPV6Open (h) and hTRPV6GEN (i).

Supplementary Fig. 4 | Density for genistein molecules and surrounding protein. a,b
Sites 1 (a) and 2 (b) of genistein viewed intracellularly along the channel pore, with molecules of genistein (yellow) and TRPV6 subunits A and C (green) as well as B and D (pink) shown in sticks and density for genistein and the surrounding protein as red and blue mesh, respectively.

Supplementary Fig. 5 | Comparison of human TRPV6 open-state structures. a-e
Open-state (apo) cryo-EM structures of full-length hTRPV6 viewed parallel to membrane in GDN detergent (a, orange), MSP2N2 nanodisc (b, green, PDB ID: 6BO8) and A8-35 amphipol (c, pink, PDB ID: 6BO9), as well as hTRPV6-CtD in GDN detergent (d, olive) and cNW11 nanodisc (e, blue). f Superposition of structures shown in a-e. g,h Parallel to membrane (g) and intracellular (h) closeup views of the pore-forming domains in structures superposed in f. Only two of four subunits are shown in g, with the front and back subunits omitted for clarity. i Parallel to membrane close-up view of S1-S4 in structures superposed in f. Supplementary Fig. 6 | Cryo-EM density for the metal ions bound at the TRPV6GEN pore intracellular entrance. a View of the intracellular entrance to the pore (grey circle in the middle), with subunits A and C colored green, B and D pink, and metal ions (M1 and M2) shown as blue spheres. The molecule of genistein at site 2 and residues surrounding and contributing to the metal binding sites are shown in sticks. b,c Close-up views of M1 (b) and M2 (c) coordinated by H582 and H587, with cryo-EM density for the histidines and metal ions shown as blue mesh and distances between them indicated (red). Fig. 7 | MD simulations of genistein binding to sites 1 and 2. a Intracellular view of genistein at the secondary position in site 2. Gray filling shows the density for genistein heavy atoms averaged over the MD runs. The non-protein cryo-EM densities for site 2 are shown as red and blue mesh (associated with genistein molecules at the primary and secondary positions, respectively). Protein subunits A/C (green) and B/D (pink) are shown as cartoon models, with residues forming hydrogen bonds and π-stacking interactions with genistein shown in sticks and labeled. The most populated MD states of genistein at the secondary position in site 2 are illustrated by representative yellow and orange stick models. b-d Measurements for four individual MD runs are shown for genistein in site 1 (b), and at the primary (c) and secondary (d) positions in site 2. Black curves show the displacement of the center of genistein molecule (center of mass of the heavy atoms in the ring structures of the molecule) relative to its initial position in each MD run (genistein position is fixed during the first 20 ns). Red and blue curves show the number of genistein-protein hydrogen bonds and π-stacking interactions, respectively.

Supplementary Fig. 8 | MD simulations of CHS binding to sites 1 and 2. a,b
Extracellular view of site 1 (a) and intracellular view of site 2 (b). Gray filling shows the density for CHS heavy atoms averaged over the MD runs. The experimental cryo-EM protein and non-protein densities are shown as blue and red mesh, respectively. Protein subunits A/C (green) and B/D (pink) are shown as cartoon models, with residues forming hydrogen bonds with CHS shown in sticks and labeled. The populated MD states of CHS are illustrated by representative yellow, orange, and magenta stick models. c,d Measurements for four individual MD runs are shown for sites 1 (c) and 2 (d). Black curves show the displacement of the center of CHS molecule (center of mass of the heavy atoms in the ring structures of the molecule) relative to its initial position in each MD run (CHS position is fixed during the first 20 ns). Red curves show the number of CHS-protein hydrogen bonds.  * In runs gen_site1_run1-4, the second genistein molecule was inserted into the primary position of site 2, but it was unstable in MD without the ligand in the secondary position.

Supplementary
** In runs gen_site2_run1-4, genistein was also inserted into site 1, but it was unstable in MD because of more extended constrictions applied to the protein (the distances were constrained between each residue in the region 563-578 for gen_site1_run1-4 and in the region 536-586 for gen_site2_run1-4). These constrictions prevented protein adaptation for the ligand binding in site 1.